Carbonic anhydrase activity in primary sensory neurons. II. Influence of environmental factors on the phenotypic expression of the enzyme in dissociated cultures of chicken dorsal root ganglion cells.

Neuronal subpopulations of dorsal root ganglion (DRG) cells in the chicken exhibit carbonic anhydrase (CA) activity. To determine whether CA activity is expressed by DRG cells maintained in in vitro cultures, dissociated DRG cells from 10-day-old chick embryos were cultured on a collagen substrate. The influence exerted by environmental factors on the enzyme expression was tested under various conditions of culture. Neuron-enriched cell cultures and mixed DRG-cell cultures (including numerous non-neuronal cells) were performed either in a defined medium or in a horse serum-supplemented medium. In all the tested conditions, subpopulations of cultured sensory neurons expressed CA activity in their cell bodies, while their neurites were rarely stained; in each case, the percentage of CA-positive neurons declined with the age of the cultures. The number and the persistence of neurons possessing CA activity as well as the intensity of the reaction were enhanced by addition of horse serum. In contrast, the expression of the neuronal CA activity was not affected by the presence of non-neuronal cells or by the rise of CO2 concentration. Thus, the appearance and disappearance of neuronal subpopulations expressing CA activity may be decisively influenced by factors contained in the horse serum. The loss of CA-positive neurons with time could result from a cell selection or from genetic repression. Analysis of the time curves does not support a preferential cell death of CA-positive neurons but suggests that the eventual conversion of CA-positive neurons into CA-negative neurons results from a loss of the enzyme activity. These results indicate that the phenotypic expression of cultured sensory neurons is dependent on defined environmental factors.

Sciatic and femoral nerve sensory neurones occupy different regions of the L4 dorsal root ganglion in the adult rat.

The topographical distribution of sciatic and femoral nerve sensory neuronal somata in the L4 dorsal root ganglion of the adult rat was mapped after retrograde tracing with one or two of the dyes Fast Blue, Fluoro-Gold, or Diamidino Yellow. The tracers were applied to the proximal transected end of either nerve alone, or from both nerves in the same animal using separate tracers. Three-dimensional reconstructions of the distribution of labelled neurones were made from serial sections of the L4 dorsal root ganglion which is the only ganglion that these two nerves share. The results showed that with little overlap, femoral nerve neurones distribute dorsally and rostrally whereas sciatic nerve neurones distribute medially and ventrally. This finding indicates the existence of a somatotopical organisation for the representation of different peripheral nerves in dorsal root ganglia of adult animals.

Modulation of the expression of the transcription factor Max in rat retinal ganglion cells by a recombinant adeno-associated viral vector

Exclusion of the transcription factor Max from the nucleus of retinal ganglion cells is an early, caspase-independent event of programmed cell death following damage to the optic axons. To test whether the loss of nuclear Max leads to a reduction in neuroprotection, we developed a procedure to overexpress Max protein in rat retinal tissue in vivo. A recombinant adeno-associated viral vector (rAAV) containing the max gene was constructed, and its efficiency was confirmed by transduction of HEK-293 cells. Retinal ganglion cells were accessed in vivo through intravitreal injections of the vector in rats. Overexpression of Max in ganglion cells was detected by immunohistochemistry at 2 weeks following rAAV injection. In retinal explants, the preparation of which causes damage to the optic axons, Max immunoreactivity was increased after 30 h in vitro, and correlated with the preservation of a healthy morphology in ganglion cells. The data show that the rAAV vector efficiently expresses Max in mammalian retinal ganglion cells, and support the hypothesis that the Max protein plays a protective role for retinal neurons.

Density, proportion, and dendritic coverage of retinal ganglion cells of the common marmoset (Callithrix jacchus jacchus)

We performed a quantitative analysis of M and P cell mosaics of the common-marmoset retina. Ganglion cells were labeled retrogradely from optic nerve deposits of Biocytin. The labeling was visualized using horseradish peroxidase (HRP) histochemistry and 3-3'diaminobenzidine as chromogen. M and P cells were morphologically similar to those found in Old- and New-World primates. Measurements were performed on well-stained cells from 4 retinas of different animals. We analyzed separate mosaics for inner and outer M and P cells at increasing distances from the fovea (2.5-9 mm of eccentricity) to estimate cell density, proportion, and dendritic coverage. M cell density decreased towards the retinal periphery in all quadrants. M cell density was higher in the nasal quadrant than in other retinal regions at similar eccentricities, reaching about 740 cells/mm² at 2.5 mm of temporal eccentricity, and representing 8-14% of all ganglion cells. P cell density increased from peripheral to more central regions, reaching about 5540 cells/mm² at 2.5 mm of temporal eccentricity. P cells represented a smaller proportion of all ganglion cells in the nasal quadrant than in other quadrants, and their numbers increased towards central retinal regions. The M cell coverage factor ranged from 5 to 12 and the P cell coverage factor ranged from 1 to 3 in the nasal quadrant and from 5 to 12 in the other quadrants. These results show that central and peripheral retinal regions differ in terms of cell class proportions and dendritic coverage, and their properties do not result from simply scaling down cell density. Therefore, differences in functional properties between central and peripheral vision should take these distinct regional retinal characteristics into account.

Caspase dependence of the death of neonatal retinal ganglion cells induced by axon damage and induction of autophagy as a survival mechanism

We examined the degeneration of post-mitotic ganglion cells in ex-vivo neonatal retinal explants following axon damage. Ultrastructural features of both apoptosis and autophagy were detected. Degenerating cells reacted with antibodies specific for activated caspase-3 or -9, consistent with the presence of caspase activity. Furthermore, peptidic inhibitors of caspase-9, -6 or -3 prevented cell death (100 µM Ac-LEDH-CHO, 50 µM Ac-VEID-CHO and 10 µM Z-DEVD-fmk, respectively). Interestingly, inhibition of autophagy by 7-10 mM 3-methyl-adenine increased the rate of cell death. Immunohistochemistry data, caspase activation and caspase inhibition data suggest that axotomy of neonatal retinal ganglion cells triggers the intrinsic apoptotic pathway, which, in turn, is counteracted by a pro-survival autophagic response, demonstrated by electron microscopy profiles and pharmacological autophagy inhibitor.

Effect of selective β-adrenoceptor blockade and surgical resection of the celiac-superior mesenteric ganglion complex on delayed liquid gastric emptying induced by dipyrone, 4-aminoantipyrine, and antipyrine in rats

There is evidence for participation of peripheral β-adrenoceptors in delayed liquid gastric emptying (GE) induced in rats by dipyrone (Dp), 4-aminoantipyrine (AA), and antipyrine (At). The present study aimed to determine whether β-adrenoceptors are involved in delayed GE induced by phenylpyrazole derivatives and the role of the prevertebral sympathetic nervous system in this condition. Male Wistar rats weighing 220-280 g were used in the study. In the first experiment rats were intravenously pretreated with vehicle (V), atenolol 30 mg/kg (ATE, β1-adrenergic antagonist), or butoxamine 25 mg/kg (BUT, β2-adrenergic antagonist). In the second experiment, rats were pretreated with V or SR59230A 2 mg/kg (SRA, β3-adrenergic antagonist). In the third experiment, rats were subjected to surgical resection of the celiac-superior mesenteric ganglion complex or to sham surgery. The groups were intravenously treated with saline (S), 240 µmol/kg Dp, AA, or At, 15 min after pretreatment with the antagonists or V and nine days after surgery. GE was determined 10 min later by measuring the percentage of gastric retention (%GR) of saline labeled with phenol red 10 min after gavage. The %GR (means±SE, n=6) values indicated that BUT abolished the effect of Dp (BUT+Dp vs V+Dp: 35.0%±5.1% vs 56.4%±2.7%) and At (BUT+At vs V+At: 33.5%±4.7% vs 52.9%±2.6%) on GE, and significantly reduced (P<0.05) the effect of AA (BUT+AA vs V+AA: 48.0%±5.0% vs 65.2%±3.8%). ATE, SRA, and sympathectomy did not modify the effects of treatments. These results suggest that β2-adrenoceptor activation occurred in delayed liquid gastric emptying induced by the phenylpyrazole derivatives dipyrone, 4-aminoantipyrine, and antipyrine. Additionally, the released neurotransmitter did not originate in the celiac-superior mesenteric ganglion complex.

Role of the ASPP Family in the Regulation of p53-Mediated Apoptotic Death of Retinal Ganglion Cells after Optic Nerve Injury

Le glaucome est la première cause de cécité irréversible à travers le monde. À présent il n’existe aucun remède au glaucome, et les thérapies adoptées sont souvent inadéquates. La perte de vision causée par le glaucome est due à la mort sélective des cellules rétiniennes ganglionnaires, les neurones qui envoient de l’information visuelle de la rétine au cerveau. Le mécanisme principal menant au dommage des cellules rétiniennes ganglionnaires lors du glaucome n’est pas bien compris, mais quelques responsables putatifs ont été proposés tels que l’excitotoxicité, le manque de neurotrophines, la compression mécanique, l’ischémie, les astrocytes réactifs et le stress oxidatif, parmis d’autres. Indépendamment de la cause, il est bien établi que la perte des cellules rétiniennes ganglionnaires lors du glaucome est causée par la mort cellulaire programmée apoptotique. Cependant, les mécanismes moléculaires précis qui déclenchent l’apoptose dans les cellules rétiniennes ganglionnaires adultes sont mal définis. Pour aborder ce point, j’ai avancé l’hypothèse centrale que l’identification de voies de signalisations moléculaires impliquées dans la mort apoptotique des cellules rétiniennes ganglionnaires offrirait des avenues thérapeutiques pour ralentir ou même prévenir la mort de celles-ci lors de neuropathies oculaires telles que le glaucome. Dans la première partie de ma thèse, j’ai caractérisé le rôle de la famille de protéines stimulatrices d’apoptose de p53 (ASPP), protéines régulatrices de la famille p53, dans la mort apoptotique des cellules rétiniennes ganglionnaires. p53 est un facteur de transcription nucléaire impliqué dans des fonctions cellulaires variant de la transcription à l’apoptose. Les membres de la famille ASPP, soit ASPP1, ASPP2 et iASPP, sont des protéines de liaison de p53 qui régulent l’apoptose. Pourtant, le rôle de la famille des ASPP dans la mort des cellules rétiniennes ganglionnaires est inconnu. ASPP1 et ASPP2 étant pro-apoptotiques, l’hypothèse de cette première étude est que la baisse ciblée de ASPP1 et ASPP2 promouvrait la survie des cellules rétiniennes ganglionnaires après une blessure du nerf optique. Nous avons utilisé un modèle expérimental bien caractérisé de mort apoptotique neuronale induite par axotomie du nerf optique chez le rat de type Sprague Dawley. Les résultats de cette étude (Wilson et al. Journal of Neuroscience, 2013) ont démontré que p53 est impliqué dans la mort apoptotique des cellules rétiniennes ganglionnaires, et qu’une baisse ciblée de ASPP1 et ASPP2 par acide ribonucléique d’interference promeut la survie des cellules rétiniennes ganglionnaires. Dans la deuxième partie de ma thèse, j’ai caractérisé le rôle d’iASPP, le membre anti-apoptotique de la famille des ASPP, dans la mort apoptotique des cellules rétiniennes ganglionnaires. L’hypothèse de cette seconde étude est que la surexpression d’iASPP promouvrait la survie des cellules rétiniennes ganglionnaires après axotomie. Mes résultats (Wilson et al. PLoS ONE, 2014) démontrent que le knockdown ciblé de iASPP exacerbe la mort apoptotique des cellules rétiniennes ganglionnaires, et que la surexpression de iASPP par virus adéno-associé promeut la survie des cellules rétiniennes ganglionnaires. En conclusion, les résultats présentés dans cette thèse contribuent à une meilleure compréhension des mécanismes régulateurs sous-jacents la perte de cellules rétiniennes ganglionnaires par apoptose et pourraient fournir des pistes pour la conception de nouvelles stratégies neuroprotectrices pour le traitement de maladies neurodégénératives telles que le glaucome.

Diabetes mellitus-related morphoquantitative changes in the celiac ganglion neurons of the dog

Diabetes mellitus is the most common endocrine disturbance of domestic carnivores and can cause autonomic neurological disorders, although these are still poorly understood in veterinary medicine. There is little information available on the quantitative adaptation mechanisms of the sympathetic ganglia during diabetes mellitus in domestic mammals. By combining morphometric methods and NADPH-diaphorase staining (as a possible marker for nitric oxide producing neurons), type I diabetes mellitus-related morphoquantitative changes were investigated in the celiac ganglion neurons in dogs. Twelve left celiac ganglia from adult female German shepherd dogs were examined: six ganglia were from non-diabetic and six from diabetic subjects. Consistent hypertrophy of the ganglia was noted in diabetic animals with increase of 55% in length, 53% in width, and 61.5% in thickness. The ordinary microstructure of the ganglia was modified leading to an uneven distribution of the ganglionic units and a more evident distribution of axon fascicles. In contrast to non-diabetic dogs, there was a lack of NADPH-diaphorase perikarial labelling in the celiac ganglion neurons of diabetic animals. The morphometric study showed that both the neuronal and nuclear sizes were significantly larger in diabetic dogs (1.3 and 1.39 times, respectively). The profile density and area fraction of NADPH-diaphorase-reactive celiac ganglion neurons were significantly larger (1.35 and 1.48 times, respectively) in non-diabetic dogs compared to NADPH-diaphorase-non-reactive celiac ganglion neurons in diabetic dogs. Although this study suggests that diabetic neuropathy is associated with neuronal hypertrophy, controversy remains over the possibility of ongoing neuronal loss and the functional interrelationship between them. It is unclear whether neuronal hypertrophy could be a compensation mechanism for a putative neuronal loss during the diabetes mellitus. (C) 2007 Elsevier Ltd. All rights reserved.

Putative antinociceptive action of nitric oxide in the caudal part of the spinal trigeminal nucleus during chronic carrageenan-induced arthritis in the rat temporomandibular joint

In order to investigate a putative role for nitric oxide (NO) in the central nociceptive processing following carrageenan-induced arthritis in the rat temporomandibular joint (TMJ), we analyzed the immunoreactivity, gene expression and activity of nitric oxide synthases (NOS) in the caudal part of the spinal trigeminal nucleus (Sp5C) during the acute (24 h), chronic (15 days) and chronic-active (14 days-24 h) arthritis. In addition, evaluation of head-withdrawal threshold was carried out in all phases of arthritis under chronic inhibition of nNOS with the selective inhibitor 7-nitroindazole (7-NI). Neurons with nNOS-like immunoreactivity (nNOS-LI) were concentrated mainly in the lamina II of the Sp5C, showing no significant statistical difference during arthritis. Only a discrete percentage of nNOS-LI neurons expressed Fos immunoreactivity. The mRNA expression for both nNOS and endothelial nitric oxide synthases (eNOS) presented no noticeable differences among the groups. No expression of inducible nitric oxide synthase (iNOS) was detected in the Sp5C by either immunohistochemistry or reverse-transcription polymerase chain reaction (RTPCR). Ca(2+)-dependent NOS activity in the ipsilateral Sp5C was significantly higher (108.3 +/- 49.2%; P<0.01) in animals during the chronic arthritis. Interestingly, this increased activity was completely abolished 24 h later, in the chronic-active arthritis. Finally, head-withdrawal threshold decreased significantly in the chronic arthritis in animals under 7-NI chronic inhibition. In conclusion, nNOS immunoreactivity and mRNA expression are stable in the Sp5C during TMJ arthritis evolution, but its activity significantly increases in the chronic-phases supporting an antinociceptive role of the nNOS as evidenced by pain threshold experiment. (C) 2009 Elsevier B.V. All rights reserved.

Participation of peripheral tachykinin NK(1) receptors in the carrageenan-induced inflammation of the rat temporomandibular joint

Temporomandibular disorders represent one of the major challenges in dentistry therapeutics. This study was undertaken to evaluate the time course of carrageenan-induced inflammation in the rat temporomandibular joint (TMJ) and to investigate the role of tachykinin NK(1) receptors. Inflammation was induced by a single intra-articular (i.art.) injection of carrageenan into the left TMJ (control group received sterile saline). Inflammatory parameters such as plasma extravasation, leukocyte influx and mechanical allodynia (measured as the head-withdrawal force threshold) and TNF alpha and IL-1 beta concentrations were measured in the TMJ lavages at selected time-points. The carrageenan-induced responses were also evaluated after treatment with the NK(1) receptor antagonist SR140333. The i.art. injection of carrageenan into the TMJ caused a time-dependent plasma extravasation associated with mechanical allodynia, and a marked neutrophil accumulation between 4 and 24 h. Treatment with SR140333 substantially inhibited the increase in plasma extravasation and leukocyte influx at 4 and 24 h, as well as the production of TNF alpha and IL-1 beta into the joint cavity, but failed to affect changes in head-withdrawal threshold. The results obtained from the present TMJ-arthritis model provide, for the first time, information regarding the time course of this experimental inflammatory process. In addition, our data show that peripheral NK(1) receptors mediate the production of both TNF alpha and IL-1 beta in the TMJ as well as some of the inflammatory signs, such as plasma extravasation and leukocyte influx, but not the nociceptive component. 2008 European Federation of Chapters of the International Association for the Study of Pain. Published by Elsevier Ltd. All rights reserved.

Macro- and microstructural organization of the rabbit's celiac-mesenteric ganglion complex (Oryctolagus cuniculus)

The macro- and microstructures of the rabbit celiac-mesenteric ganglion complex are described in 20 young animals. We found ten celiac ganglia, twenty-seven cranial mesenteric ganglia and eleven celiac-mesenteric ganglia. The celiac ganglia had a rectangular shape in nine cases (90%) and a circular one in one case (10%). The cranial mesenteric ganglia presented triangular (66.7%), rectangular (11.1%), L-shape (18.5%) and semilunar (3.7%) arrangements. The celiac-mesenteric ganglia were organized in three patterns: a single left celiac-mesenteric ganglion having a caudal portion (72.7%); celiac-mesenteric ganglia without a caudal portion (18.2%) and a single celiac-mesenteric ganglion with two portions: left and right (9.1%).The microstructure was investigated in nine celiac-mesenteric ganglia. The results showed that the celiac-mesenteric ganglion is actually a ganglion complex constituted of an agglomerate of ganglionic units separated by nerve fibers, capillaries and septa of connective tissue. Using the semi-thin section method we described the cellular organization of the celiac-mesenteric ganglion complex. Inside of each ganglionic unit, there were various cell types: principal ganglion neurons (PGN), glial cells (satellite cells) and SIF cells (small intensely fluorescent cells or small granular cells), which are the cytologic basis for each ganglionic unit of the rabbit's celiac-mesenteric ganglion complex.

Putative antinociceptive action of nitric oxide in the caudal part of the spinal trigeminal nucleus during chronic carrageenan-induced arthritis in the rat temporomandibular joint

In order to investigate a putative role for nitric oxide (NO) in the central nociceptive processing following carrageenan-induced arthritis in the rat temporomandibular joint (TMJ), we analyzed the immunoreactivity, gene expression and activity of nitric oxide synthases (NOS) in the caudal part of the spinal trigeminal nucleus (Sp5C) during the acute (24 h), chronic (15 days) and chronic-active (14 days-24 h) arthritis. In addition, evaluation of head-withdrawal threshold was carried out in all phases of arthritis under chronic inhibition of nNOS with the selective inhibitor 7-nitroindazole (7-NI). Neurons with nNOS-like immunoreactivity (nNOS-LI) were concentrated mainly in the lamina II of the Sp5C, showing no significant statistical difference during arthritis. Only a discrete percentage of nNOS-LI neurons expressed Fos immunoreactivity. The mRNA expression for both nNOS and endothelial nitric oxide synthases (eNOS) presented no noticeable differences among the groups. No expression of inducible nitric oxide synthase (iNOS) was detected in the Sp5C by either immunohistochemistry or reverse-transcription polymerase chain reaction (RTPCR). Ca(2+)-dependent NOS activity in the ipsilateral Sp5C was significantly higher (108.3 +/- 49.2%; P<0.01) in animals during the chronic arthritis. Interestingly, this increased activity was completely abolished 24 h later, in the chronic-active arthritis. Finally, head-withdrawal threshold decreased significantly in the chronic arthritis in animals under 7-NI chronic inhibition. In conclusion, nNOS immunoreactivity and mRNA expression are stable in the Sp5C during TMJ arthritis evolution, but its activity significantly increases in the chronic-phases supporting an antinociceptive role of the nNOS as evidenced by pain threshold experiment. (C) 2009 Elsevier B.V. All rights reserved.

Phenotypic alterations of neuropeptide Y and calcitonin gene-related peptide-containing neurons innervating the rat temporomandibular joint during carrageenan-induced arthritis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Macro- and microstructure of the superior cervical ganglion in dogs, cats and horses during maturation

The superior cervical ganglion (SCG) provides sympathetic input to the head and neck, its relation with mandible, submandibular glands, eyes (second and third order control) and pineal gland being demonstrated in laboratory animals. In addition, the SCG's role in some neuropathies can be clearly seen in Horner's syndrome. In spite of several studies published involving rats and mice, there is little morphological descriptive and comparative data of SCG from large mammals. Thus, we investigated the SCG's macro- and microstructural organization in medium (dogs and cats) and large animals (horses) during a very specific period of the post-natal development, namely maturation (from young to adults). The SCG of dogs, cats and horses were spindle shaped and located deeply into the bifurcation of the common carotid artery, close to the distal vagus ganglion and more related to the internal carotid artery in dogs and horses, and to the occipital artery in cats. As to macromorphometrical data, that is ganglion length, there was a 23.6% increase from young to adult dogs, a 1.8% increase from young to adult cats and finally a 34% increase from young to adult horses. Histologically, the SCG's microstructure was quite similar between young and adult animals and among the 3 species. The SCG was divided into distinct compartments (ganglion units) by capsular septa of connective tissue. Inside each ganglion unit the most prominent cellular elements were ganglion neurons, glial cells and small intensely fluorescent cells, comprising the ganglion's morphological triad. Given this morphological arrangement, that is a summation of all ganglion units, SCG from dogs, cats and horses are better characterized as a ganglion complex rather than following the classical ganglion concept. During maturation (from young to adults) there was a 32.7% increase in the SCG's connective capsule in dogs, a 25.8% increase in cats and a 33.2% increase in horses. There was an age-related increase in the neuronal profile size in the SCG from young to adult animals, that is a 1.6-fold, 1.9-fold and 1.6-fold increase in dogs, cats and horses, respectively. on the other hand, there was an age-related decrease in the nuclear profile size of SCG neurons from young to adult animals (0.9-fold, 0.7-fold and 0.8-fold in dogs, cats and horses, respectively). Ganglion connective capsule is composed of 2 or 3 layers of collagen fibres in juxtaposition and, as observed in light microscopy and independently of the animal's age, ganglion neurons were organised in ganglionic units containing the same morphological triad seen in light microscopy. Copyright (c) 2007 S. Karger AG, Basel.